ASBTRACT. Arginylation mediated by arginyltransferase (ATE1) is a posttranslational modification that plays a global role in mammals. Arginylation is essential for mammalian embryogenesis and regulation of the cytoskeleton function in cell migration, a process that plays key roles during tissue morphogenesis and cancer metastases. Arginylation occurs through formation of arginine linkage to E or D amino acid residues that are located either internally or at the N-terminus of target proteins. Addition of N-terminal Arg to proteins may induce their ubiquitination and degradation by the N-end rule pathway, however recent studies suggest that the scope of the biological effects of arginylation is much broader, reminiscent of the effects of protein phosphorylation. A recent explosion of studies has put arginylation on the map of intracellular metabolic pathways and biological processes and demonstrated its key involvement in the central events of cell metabolism, normal function of multiple physiological systems, and cell transition to metastatic cancers. Research in the protein arginylation field is however hampered by the lack of antibodies or other tools that selectively recognize arginylated proteins. To address this issue we propose utilizing our combined expertise in generating arginylated linkage peptides and yeast genetics to generate a diverse array of antibodies that will advance the field of cell biology. In this application, we propose to use innovative yeast display of a highly diverse human antibody Fab library with self-diversifying ability to select for arginylation specific binders. We will employ synthetic peptides which encompass both the isopeptide bond and the sequence surrounding the arginine attachment sites in ?- synuclein (Phase I) and will expand this approach to include the development of arginylation antibodies of global specificity, which can serve as a major tools for biomedical research including inverse correlation of arginylation and ageing, neurodegeneration and clinical diagnostics (Phase II). As the result of this project, a set of site-specific arginylation and pan-arginylation antibodies will be developed. The scientific community will use these tools to chart new pathways in cell biology and biochemistry. These tools will be used as detection agents in protein chemistry, clinical diagnostic applications and imaging studies. These tools will unmask arginylation of cellular proteins and uncover new roles unique to arginylated proteins. Given the poor nature of tools available for the arginylation field, as a proof-of- principle, the phase I grant will focus on E46- and E83- linkages in -synuclein and subsequently in Phase II expand to a range of arginylated protein targets with side chain and N-terminal linkages. We believe that development of arginine linkage selective tools will have as great an impact on cell biology as the development of phospho-specific antibodies had on cellular phosphorylation.